Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The diploid chromosome complement of the domestic dog Canis familiaris consists of 76 acrocentric autosomes and an X-Y sex chromosome pair (Selden et al., 1975). The autosomes, in addition to being numerous, are fairly small and resemble each other even after G-banding. The technical difficulties of working on the canine autosomes due to their small size, combined with the evolutionary history of the canine karyotype as a highly derived version of the primitive carnivore karyotype (probably as a result of Robertsonian fusions--Wayne et al., 1987b), have resulted in an almost complete lack of knowledge about the comparative cytogenetics of the dog. Efforts to perform positional cloning of mutations using canine models of gene defects are seriously hampered without a knowledge of chromosomal synteny relationships between the dog and species with detailed chromosomal maps (such as man and mouse).
Previous techniques in which marking of canine autosomes have been attempted include G-banding, in situ hybridization of highly repetitive DNAs, C-banding, N-banding, and Q-banding.
Because of the small size and numerous amount of canine autosomes, two studies on the G-banded karyotype of the dog differed on the banding patterns and numbering systems for several of the autosomes (Selden et al., 1975; Manolache et al., 1976). Later reports attempted to resolve some of the inconsistencies by relying upon G-banded karyotypes from sequentially stained amethopterin synchronized lymphocyte cultures (Stone et al., 1991) or by using fibroblast cell lines of the grey wolf Canis lupus (a species with a completely homologous karyotype to the domestic dog--Wayne et al., 1987a).
The in situ hybridization studies that have been carried out on canine autosomes have been limited in scope to grain count statistics on the binding of canine satellite DNAs to the centromeric heterochromatin (Modi et al., 1988).
C-banding of the centromeric heterochromatin labels only 7 or 8 unspecified autosomes, while N-banding (silver staining indicative of nucleolar organizer regions) marked three pairs of autosomes and the Y chromosome (Pathak et al., 1982; Stone et al., 1991). Q-banding resulted in fluorescing regions which corresponded to darkly staining G-band regions, but the resolution was minimal (Stone et al., 1991).
Despite these various attempts to mark canine autosomes for identification and/or differentiation, the problems associated with the small size and numerous amounts of the canine autosomes has still hampered the development of an accurate and reproducible method for identification and/or differentiation thereof.
The development of identification methods for the canine autosomes that will permit rapid and unequivocal recognition of chromosome groups is an important first step in the comparative mapping of the canine genome. Further, these methods will ideally be capable of being applied simultaneously with the in situ hybridization of unique copy euchromatic chromosomal probes of interest from dog or other species (such as man or mouse).
In mammalian species other than man and mouse, the usefulness of the different types of repetitive probes for chromosome banding and recognition studies is difficult to predict. For example, fluorescent in situ hybridizations of Alu-like sequences to chromosome sets from pig, sheep, and cow were found to produce indistinct and diffuse hybridization patterns that were not useful for chromosome recognition (Yasue et al., 1991; Rajcan-Separovic and Sabour, 1993). By contrast, the highly repetitive C5 satellite probe from the Chinese muntjac which was found to hybridize to the centromeric heterochromatin of the Chinese muntjac and the Indian muntjac was shown to also bind to a limited number (ca. 30) of interstitial sites on the arms of only the Indian muntjac chromosomes (Lin et al., 1991).
For the dog, previous work on interspersed repetitive DNAs has been limited to the identification of a LINE element homologous to the Kpn repeat of primates (Katzir et al., 1985; Amariglio et al., 1991) and the canid-specific SINE element (Minnick et al., 1992).
It should be apparent from the above discussion that the accuracy of particular chromosome marking methods can vary between species. A successful G-banding in one species, or a successful FISH technique in a species, does not indicate that G-banding and/or FISH techniques will be successful in identifying and/or differentiating chromosomes from another different species. In view of the problems encountered in marking canine chromosomes, despite some success in marking chromosomes from other species, a need continues to exist for an accurate method for differentiating and/or identifying canine chromosomes.